JP7032775B2 - How to streamline the expression of artificially synthesized mRNA - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Reason for publication 1 Website publication date: July 4, 2016 Website address: http: // shibu. pharm. or. jp / tokai / 160709Program. pdf (2) Reason for publication 2 Publisher: Pharmaceutical Society of Japan Tokai Branch Publication name: 62nd (2016) Pharmaceutical Society of Japan Tokai Branch General Assembly / Meeting Abstracts Publication date: July 9, 2016 ( 3) Reasons for disclosure 3 Meeting name: 62nd (2016) Pharmaceutical Society of Japan Tokai Branch General Assembly / Meeting Date: July 9, 2016

The present invention relates to a technique for improving the expression efficiency of artificially synthesized mRNA in a target cell and its use.

Until now, gene therapy has been carried out using DNA such as viruses as a vector, but the risk of carcinogenesis due to incorporation into the genome remains a major problem. On the other hand, artificially synthesized mRNA is attracting attention as a safe nucleic acid drug that does not have a risk of insertion into the genome unlike DNA, but it has been pointed out that RNA has inherent instability and low translation efficiency. ing.

The research group of the present inventors has been developing a technique for stabilizing artificially synthesized mRNA through elucidation of the intracellular degradation mechanism of exogenous synthetic mRNA (artificial synthetic mRNA). Conventionally, it has been believed that artificially synthesized mRNA is degraded by the same molecular mechanism as intracellular mRNA (endogenous mRNA), but according to our research, artificially synthesized mRNA is degraded by a completely different mechanism. We succeeded in stabilizing artificially synthesized mRNA by inhibiting the degradation system (Patent Document 1).

JP-A-2015-226531

Looking ahead to the future development of RNA drugs, it is desired to further increase the expression level of artificially synthesized mRNA through intracellular stabilization or improvement of translation efficiency. That is, there is still a high need for a technique for improving the expression efficiency of artificially synthesized mRNA. Therefore, it is an object of the present invention to provide a technique for improving the expression efficiency of artificially synthesized mRNA in a target cell and its use.

While studying to solve the above problems, the present inventors considered that there must be a mechanism for recognizing the artificially synthesized mRNA that has invaded the cell as exogenous and distinguishing it from the intracellular mRNA, and considered that the idea was I proceeded with the research below. As a result of formulating a detailed and detailed research plan and conducting various experiments, the identification mechanism of exogenous mRNA was clarified, and OAS (2'-5'oligoadenylate synthetase) was identified as a factor involved in the identification of mRNA in cells. I succeeded in doing it. OAS does not bind to intracellular mRNA, but selectively binds to and identifies exogenous synthetic mRNA. Surprisingly, it was found that inhibition of this factor not only stabilizes exogenous synthetic mRNA, but also increases its translation rate. That is, it was found that inhibition of OAS can improve the expression efficiency of exogenous synthetic mRNA (artificial synthetic mRNA) by stabilizing mRNA and improving translation efficiency. Inhibition of OAS targets the process of distinguishing synthetic mRNA from extrinsic upstream of the degradation system, and is distinct from the means for inhibiting the degradation system of exogenous synthetic mRNA itself (Patent Document 1). do. In addition, it can be said to be a more effective and effective means in that it not only stabilizes synthetic mRNA but also causes an increase in the amount of translation. The following inventions are mainly based on the above achievements and considerations.
[1] The mRNA of the target gene and
2'-5'-combined with a function-suppressing substance of oligoadenylic acid synthase,
The function-suppressing substance is a compound selected from the group consisting of the following (a) to (d) or a 2', 5'-oligoadenylic acid derivative.
The 2'-5'-oligoadenylic acid synthase is 2'-5'-oligoadenylate synthase 3.
Composition for expressing the target gene in the target cell:
(a) siRNA targeting 2'-5'-oligoadenylate synthase;
(b) 2'-5'-Nucleic acid construct that produces siRNA targeting oligoadenylate synthase in cells;
(c) Antisense nucleic acid targeting 2'-5'-oligoadenylate synthase;
(d) Ribozyme targeting 2'-5'-oligoadenylic acid synthase.
[2] The composition according to [1], which is a kit comprising a first element containing the mRNA and a second element containing the function-suppressing substance.
[3] The composition according to [1], which contains the mRNA and the function-suppressing substance.
[4] The composition according to [1], which contains the mRNA and is characterized in that the function-suppressing substance is introduced in combination at the time of introduction into a target cell.
[5] The composition according to [1], which contains the function-suppressing substance and is characterized in that the mRNA is co-introduced at the time of introduction into a target cell.
[6] The composition according to any one of [1] to [5], wherein the mRNA comprises a 5'cap structure, a Kozak sequence, a coding region of the target gene, and a poly (A) chain.
[7] The composition according to [6], wherein the mRNA further comprises a stabilized cis sequence located between the coding region and the poly (A) chain.
[8] The composition according to any one of [1] to [7], wherein the gene is an enzyme gene.
[9] The composition according to [8], wherein the enzyme is a nuclease selected from the group consisting of ZFN, TALEN and CRISPR-Cas9.
[10] A hepatitis B virus removing agent containing the composition according to [9] as an active ingredient.
[11] A method for expressing a target protein in a target cell, which comprises only the following steps (1) and (2).
(1) Step of introducing 2'-5'-oligoadenylate synthase function-suppressing substance into target cells;
(2) Step of introducing mRNA of target gene into the target cell;
The function-suppressing substance is a compound selected from the group consisting of the following (a) to (d) or a 2', 5'-oligoadenylic acid derivative.
The 2'-5'-oligoadenylate synthase is 2'-5'-oligoadenylate synthase 3, method:
(a) siRNA targeting 2'-5'-oligoadenylate synthase;
(b) 2'-5'-Nucleic acid construct that produces siRNA targeting oligoadenylate synthase in cells;
(c) Antisense nucleic acid targeting 2'-5'-oligoadenylate synthase;
(d) Ribozyme targeting 2'-5'-oligoadenylic acid synthase.
[12] The method according to [11], wherein the target cell is a cell separated from a living body.
In addition, the present invention can also be realized in the following forms.

[1] The mRNA of the target gene and
2'-5'-combined with a function inhibitor of oligoadenylate synthase,
A composition for expressing a target gene in a target cell.
[2] The composition according to [1], which is a kit comprising a first element containing the mRNA and a second element containing the function-suppressing substance.
[3] The composition according to [1], which contains the mRNA and the function-suppressing substance.
[4] The composition according to [1], which contains the mRNA and is characterized in that the function-suppressing substance is introduced in combination at the time of introduction into a target cell.
[5] The composition according to [1], which contains the function-suppressing substance and is characterized in that the mRNA is co-introduced at the time of introduction into a target cell.
[6] The composition according to any one of [1] to [5], wherein the mRNA comprises a 5'cap structure, a Kozak sequence, a coding region of the target gene, and a poly (A) chain.
[7] The composition according to [6], wherein the mRNA further comprises a stabilized cis sequence located between the coding region and the poly (A) chain.
[8] The composition according to any one of [1] to [7], wherein the function-suppressing substance is a compound selected from the group consisting of the following (a) to (d):
(a) siRNA targeting 2'-5'-oligoadenylate synthase;
(b) 2'-5'-Nucleic acid construct that produces siRNA targeting oligoadenylate synthase in cells;
(c) Antisense nucleic acid targeting 2'-5'-oligoadenylate synthase;
(d) Ribozyme targeting 2'-5'-oligoadenylic acid synthase.
[9] The composition according to any one of [1] to [7], wherein the function-suppressing substance is a 2', 5'-oligoadenylic acid derivative.
[10] The composition according to any one of [1] to [9], wherein the 2'-5'-oligoadenylate synthase is 2'-5'-oligoadenylate synthase 3.
[11] The composition according to any one of [1] to [10], wherein the gene is an enzyme gene.
[12] The composition according to [11], wherein the enzyme is a nuclease selected from the group consisting of ZFN, TALEN and CRISPR-Cas9.
[13] A hepatitis B virus removing agent containing the composition according to [12] as an active ingredient.
[14] A method for expressing a target protein in a target cell, which comprises the following steps (1) and (2):
(1) Step of introducing 2'-5'-oligoadenylate synthase function-suppressing substance into target cells;
(2) A step of introducing mRNA of a target gene into the target cell.
[15] The method according to [14], wherein the 2'-5'-oligoadenylate synthase is 2'-5'-oligoadenylate synthase 3.
[16] The method according to [14] or [15], wherein the target cell is a cell separated from a living body.

Luciferase siRNA was introduced into HeLa cells as OAS1 siRNA, OAS2 siRNA, OAS3 siRNA or control siRNA. 48 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, 3 hours later, the cells were washed with PBS and 0, 1, 2, 4 hours later, the cells were collected. A: The results show the detection of 5xFlag-EGFPpA72 mRNA (upper panel) by the Northern blot method and 28S rRNA (lower panel) by ethidium bromide. B: Shows the result of quantifying A. The amount of mRNA after 0 hours is shown as 100%. C: The result of calculating the half-life of mRNA is shown. Luciferase siRNA was introduced into HeLa cells as OAS1 siRNA, OAS2 siRNA, OAS3 siRNA or control siRNA. 48 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, 6 hours later, the cells were washed with PBS, and 7 hours later, the cells were collected. A: Western blotting was used to verify the expression of 5xFlag-EGFP. The results of Western blotting using anti-Flag antibody (upper panel) and anti-GAPDH antibody (lower panel) are shown. B: Shows the result of quantifying A. The amount of 5xFlag-EGFP is standardized by the amount of GAPDH, and is shown as a relative value with the amount of 5xFlag-EGFP in the control as 1. A: pCMV-5xMyc-OAS3, pCMV-5xMyc-PABPC1 or pCMV-5xMyc as a control was introduced into HeLa cells. 24 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, and 3 hours after that, cells were collected. Expression of 5xFlag-EGFPpA72 mRNA (upper panel) was confirmed by Northern blotting, and expression of 5xMyc-OAS3 and 5xMyc-PABPC1 proteins was confirmed by Western blotting. RNA and protein of the solubilized fraction were detected in lanes 1 to 3 and the immunoprecipitated fraction was detected in lanes 4 to 6. HeLa cells were introduced with pCMV-5xMyc-Dom34 along with pCMV-5xFlag-RNaseL, pCMV-5xFlag-RNaseL Y312A or pCMV-5xFlag as a control. Cells were harvested 24 hours after introduction. A: The results of Western blotting using anti-Myc antibody (upper panel) and anti-Flag antibody (lower panel) are shown. Proteins in the solubilized fraction were detected in lanes 1 to 3, and the proteins in the immunoprecipitated fraction were detected in lanes 4 to 6. B: Shows the result of quantifying A. The amount of 5xMyc-Dom34 is standardized with the amount of 5xFlag-RNaseL or 5xFlag-RNaseL Y312A, and the amount of 5xMyc-Dom34 co-precipitated with 5xFlag-RNaseL is shown as 100%. The figure which shows the target position of siRNA prepared for OAS1, OAS2, OAS3. Degradation mechanism of exogenous mRNA (artificial synthetic mRNA). Structure of mRNA (5xFlag-EGFP-pA72 mRNA) used in the experiment (base sequence: SEQ ID NO: 1, encoding amino acid sequence: SEQ ID NO: 2). Vector-derived sequence (SEQ ID NO: 3), Kozak sequence (CCCACC), coding region (SEQ ID NO: 4), stop codon, restriction enzyme sites (EcoRI, EcoRV, SalI) (SEQ ID NO: 3) from the 5'end to the 3'end. 5), β-globin 3'untranslated region (SEQ ID NO: 6), β-globin gene genome sequence (AATGATG) linked to 3'untranslated region, restriction enzyme site (XbaI) (TCTAGA), poly (A) chain Is placed.

1. 1. Composition for Expression of Target Gene The first aspect of the present invention relates to a composition for expressing the target gene in a target cell (hereinafter, also referred to as “composition of the present invention”). Through our studies, we have elucidated the mechanism by which synthetic mRNAs that have invaded cells are recognized as exogenous and are distinguished from intracellular mRNAs, and the overall picture of the degradation pathway of artificially synthesized mRNAs in cells has been clarified. .. In addition, by inhibiting 2'-5'-oligoadenylic acid synthase (OAS), which is the central molecule of the successful identification mechanism, artificially synthesized mRNA is stabilized and its translation amount is increased, and the artificially synthesized mRNA is increased. It was experimentally confirmed that the expression of is more efficient. That is, it was found that the expression of artificially synthesized mRNA in the target cell can be made more efficient by introducing a substance that suppresses the function of OAS when introducing the artificially synthesized mRNA into the target cell. rice field. Based on this finding, the composition of the present invention has two elements, that is, the mRNA of the target gene (hereinafter, also referred to as "mRNA of the present invention") and the function-suppressing substance of OAS (hereinafter, "function-suppressing substance of the present invention"). It is also characterized by the fact that it is used in combination. In the present specification, "using ... in combination" or "combining ..." means that the above two elements are used in combination. Specific aspects of the combined use will be described below. The mRNA of the present invention may be referred to as "artificial synthetic mRNA" for the purpose of expressing the feature that it is prepared by artificial manipulation and distinguishing it from intracellular (intrinsic) mRNA. Also, the two terms "suppression" and "inhibition" have overlapping meanings and are often used interchangeably. Therefore, in the present specification, the term "suppression" is used in a unified manner unless it is particularly necessary to distinguish it from the contexts before and after.

"Efficiency of mRNA expression" means that mRNA is highly expressed, that is, the amount of expression product (protein) is increased. Efficiency of expression can be achieved by stabilizing mRNA and / or improving translation efficiency. Typically, according to the present invention, the expression rate of mRNA is increased by both stabilizing mRNA and improving translation efficiency (increasing the amount of translation).

In one aspect, the composition of the present invention is provided in the form of a kit comprising a first element containing the mRNA of the present invention and a second element containing the function-suppressing substance of the present invention. In this case, both elements will be administered to the target cells simultaneously or at predetermined time intervals. Preferably, the first element is introduced with a predetermined time difference after the second element is introduced in consideration of the time required for the onset of the action of the function-suppressing substance. For example, the first element is introduced 1 hour to 48 hours, preferably 4 hours to 30 hours after the introduction of the second element. The first element and the second element may be introduced at the same time. "Simultaneity" here does not require strict simultaneity. Therefore, not only when the introduction of both elements is carried out under the condition that there is no time difference, such as introduction into the target cell after mixing both elements, but also the introduction and administration of the other immediately after the introduction of one of the two elements, etc. The concept of "simultaneous" here also includes the case where the introduction of the elements is carried out under conditions where there is virtually no time lag.

In another aspect of the present invention, the composition of the present invention will be provided as a compounding agent in which the first element and the second element are mixed. On the other hand, the composition may contain the mRNA of the present invention, and the function-suppressing substance of the present invention may be introduced in combination at the time of introduction thereof. The timing of introduction of the composition and the function-suppressing substance in this case is the same as in the case of the above-mentioned kit form. That is, preferably, after introducing the function-suppressing substance, the composition containing the mRNA of the present invention is introduced. Further, contrary to the above aspect, a composition containing a function-suppressing substance may be prepared, and the mRNA of the present invention may be introduced in combination at the time of introduction thereof. The timing of introduction in this case conforms to the case of the above embodiment.

Exonuclease inhibitors, endonuclease inhibitors, phospholipids, calcium phosphate, polyethyleneimine, nanomicellar forming agents, polyethylene glycol-polycations, buffers, inorganic salts, 2 The composition of the present invention comprises valent ions and the like, antibiotics and the like for the purpose of preventing contamination with bacteria, and animal serum, growth factors, sugars, vitamins, divalent ions and the like for the purpose of enhancing the proliferation ability of cells. May be contained in. In addition, other components that are acceptable in the formulation (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, saline, etc.) It may be contained in the composition of the present invention. Further, a special synthetic medium such as Opti-MEM provided by Life Technologies may be used for the purpose of enhancing the cell introduction efficiency of the active ingredient.

2. 2. MRNA of target gene
The mRNA (mRNA of the present invention) which is a component of the composition of the present invention comprises a coding region of a target gene (a region encoding a protein which is an expression product of the target gene). The "target gene" is a gene expressed in a target cell using the mRNA of the present invention. Various genes can be adopted as target genes. Examples of genes of interest include genes such as enzymes (eg, nucleases (ZFN (Zinc Finger Nuclease), TALEN (Transcription Activator-Like Effector Nuclease), CRISPR-Cas9, etc.), cytokines, hormones, neurotransmitters, etc., and their functional decline (eg,). Genes that cause diseases such as (due to mutations) and deficiencies, genes that are functioning normally but whose expression is desired to be enhanced, and genes that target cells do not originally have and are targeted by their expression. Genes that are beneficial for cell survival, maintenance, etc., genes that act on target cells to enhance the functions that the target cells originally have, or genes that encode proteins that exert functions different from the functions that the target cells originally have, for target cells It is a gene that encodes a protein that does not act and is secreted from the target cell and acts on surrounding cells (for example, a protein involved in an intercellular network). It does not substantially act on the target cell and surrounding cells. A gene encoding a protein can also be a target gene. As such a gene, for example, a gene encoding a protein used in a pharmaceutical product or the like (for example, a human erythropoietin gene, a human fibrinogen gene, a human serum albumin gene, a human lactoferrin gene, etc. A human α-glucosidase gene) can be mentioned. By adopting such a gene, it is possible to produce a recombinant protein that can be used as a drug or the like in a target cell. The ZFN gene (IL28B is targeted). The sequence of the TALEN gene is in SEQ ID NO: 7 (ZFN-right) and SEQ ID NO: 8 (ZFN-left), the sequence of the TALEN gene is in SEQ ID NO: 9, and the sequence of the CRISPR-Cas9 gene (for example, Science. 2013 Feb 15; 339 (). 6121): 819-23. Can be referred to) are shown in SEQ ID NO: 10, respectively.

A "target cell" is a cell in which a target gene is expressed. In the present invention, the OAS function-suppressing substance and the mRNA of the present invention are introduced into the target cell. The target cell is not particularly limited, and various eukaryotic cells can be used as the target cell. For example, as target cells, various cells of mammals (human, monkey, bovine, horse, rabbit, mouse, rat, guinea pig, hamster, etc.) such as myocardial cells, smooth muscle cells, fat cells, fibroblasts, bone cells, etc. Cartilage cells, osteoclasty cells, parenchymal cells, epidermal keratinized cells (keratinocytes), epithelial cells (skin epidermal cells, corneal epithelial cells, conjunctival epithelial cells, oral mucosal epithelium, hair follicle epithelial cells, oral mucosal epithelial cells, airway mucosal epithelium Cells, intestinal mucosal epithelial cells, etc.), endothelial cells (corneal endothelial cells, vascular endothelial cells, etc.), nerve cells, glial cells, splenocytes, pancreatic β cells, mesangium cells, Langerhans cells, hepatocytes, precursor cells or stem cells thereof. Or, use artificial pluripotent stem cells (iPS cells), mesenchymal stem cells (MSC), embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonic tumor cells (EC cells), etc. Can be done. In addition, passage cells, cells induced to differentiate into a specific cell lineage, established cells (eg, HeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, COS-7 cells, NIH3T3 cells, Sf9 cells). Can also be used.

The present invention is applied to a target cell isolated from a living body (that is, an isolated target cell) or a target cell constituting the living body. Therefore, it is possible to carry out the present invention in any of in vitro, in vivo and ex vivo environments. Here, "isolated" means that it is in a state of being taken out from its original environment (for example, a state constituting a living body). Therefore, usually, the isolated target cells are present in a culture vessel or a storage vessel and can be artificially manipulated in vitro. Specifically, cells isolated from the living body and in a cultured state outside the living body (including established cells) are eligible as isolated target cells. In addition, as long as it is in the isolated state in the above sense, it is an isolated cell even in the state where the tissue is formed.

The isolated target cells can be prepared from a living body (for example, a patient). On the other hand, cells obtained from RIKEN BioResource Center, National Institute of Technology and Evaluation, ATCC (American Type Culture Collection), DSMZ (German Collection of Microorganisms and Cell Cultures), etc. are isolated targets. It can also be used as a cell.

The mRNA of the present invention has a 5'cap structure (a structure in which m ⁷ G (7-methylguanosine) is bound to a 5'terminal nucleoside via a 5'-5'triphosphate bridge) and poly (A) required for its translation. ) Also has a chain. The length of the poly (A) chain is not particularly limited, but is, for example, 30 to 200 bases. The translation initiation factor eIF4E binds to the 5'cap structure, and the poly (A) -binding protein PABP (poly (A) -binding protein) binds to the poly (A) chain, both of which are scaffold proteins. By forming a complex via, the mRNA has a cyclic structure (Wells SE, et al. Mol Cell. 1998; 2: 135-140). In addition, the translation terminator eRF3 forms a complex with PABP-eIF4G and loops out the 3'UTR (Uchida N, et al. J Biol Chem. 2002; 277: 50286-50292). Such mRNA cyclization physically brings the translation termination site and the translation initiation site closer to each other, and the ribosome that has completed translation is recycled from the stop codon to the next translation initiation without going through the 3'UTR. Greatly contributes to the efficiency of the start. The 5'end cap structure and the 3'end poly (A) chain not only improve the efficiency of such translation, but also stabilize the mRNA by inhibiting the degradation of mRNA from the end by exonuclease, which improves the efficiency of translation. It greatly contributes to the regulation of gene expression after transcription in both processes of mRNA stabilization.

In addition to the above elements, the mRNA of the present invention may include a 5'end untranslated region (5'UTR) and / or a 3'end untranslated region (3'UTR). The Kozak consensus is usually used as part of the 5'UTR. Also, preferably, a stabilized cis sequence is used as part of the 3'UTR. Further stabilization of mRNA can be achieved by using the stabilized cis sequence. A specific example of a stabilized cis sequence is PRE (pyrimidine-rich element). For example, by interposing β-globin PRE between the coding region and the poly (A) chain to construct mRNA, more stable mRNA can be obtained. The sequence of the PRE of β-globin is shown in SEQ ID NOs: 11 to 13 in the sequence listing (see Mol Cell Biol. Sep 2001; 21 (17): 5879-5888). The mRNA of the present invention may be used in whole or in part (however, containing PRE) of the 3'UTR (specific example shown in SEQ ID NO: 6) of the gene containing PRE in its 3'UTR such as β-globin gene. 3'UTR may be configured. On the other hand, in contrast to stabilized cis sequences, cis factors such as ARE (AU-rich element) and GRE (GU-rich element) are known to destabilize mRNA (eg, Louis I et al. , 2014, J Interferon Cytokine Res. 34: 233-241). Therefore, the mRNA of the present invention preferably does not contain a cis sequence that causes such destabilization.

As elements that can constitute 5'UTR or 3'UTR of the mRNA of the present invention, in addition to the above elements, a sequence derived from a template (commercially available cloning plasmid, etc.) used for mRNA preparation, a restriction enzyme recognition sequence ( Restriction enzyme site) can be mentioned.

The mRNA of the present invention can be prepared by a method such as an in vitro transcription system or chemical synthesis. Using a kit for in vitro transcription (for example, RiboMAX system provided by Promega, CUGA7 in vitro transcription kit provided by Nippon Gene, MEGAscript T7 kit provided by Life Technologies) makes it easy to prepare the desired mRNA. It is possible. The addition of the 5'cap structure may be performed by a known method, for example, 3'-O-Me-m7G (5') ppp (5') G RNA Cap Structure Analog provided by New England Biolabs can be used. ..

As the mRNA of the present invention, two or more types of mRNA may be used in combination. For example, an mRNA having a region encoding a specific gene and an mRNA having a region encoding an expression product that interacts with the expression product of the gene can be used in combination.

The amount of mRNA used in the composition of the present invention is such that a sufficient amount of expression product is contained in the target cell while considering the purpose of use of the composition of the present invention, the characteristics of the target gene to be used, the type of target cell, and the like. It may be set so that it can be obtained. To give an example of the amount of mRNA, it is advisable to contain 0.5 to 1.0 μg of mRNA per 3 cm culture dish as a single dose.

3. 3. Function-suppressing substance The function-suppressing substance in the present invention acts on 2'-5'-oligoadenylic acid synthase (OAS) and suppresses or inhibits the discrimination of artificially synthesized mRNA by OAS. That is, the target of the function-suppressing substance of the present invention is OAS. It is known that there are roughly three types of OAS, OAS1, OAS2, and OAS3. The function-suppressing substance exerts an inhibitory effect on at least one of these molecules. Here, in the mechanism of artificially synthesized mRNA degradation revealed by the present inventors (see FIG. 6), (i) artificially synthesized mRNA is bound and identified by oligoadenylate synthase OAS3 when introduced into the cytoplasm. .. (ii) OAS3 bound to this artificially synthesized mRNA synthesizes 2', 5'-oligoadenylic acid (2-5A) using ATP as a substrate. (iii) 2-5A binds to the endonuclease RNase L and converts the inactive monomer to the active dimer. (iv) On the other hand, artificially synthesized mRNA is translated by ribosomes in the cytoplasm, but unlike intracellular mRNA, exogenous artificially synthesized mRNA, which cannot form mRNP, has low translation efficiency and easily forms a secondary structure. , Ribosome stalls on the way. (v) Dom34-GTPBP2 associates with the stalled ribosome and specifically recruits RNase L, which has become an active dimer. (vi) As a result, exogenous artificially synthesized mRNA is specifically degraded by RNase L.

OAS3, the largest of the OAS, has multiple functional domains and has the characteristic of recognizing longer RNA, and plays a central role in the identification of exogenous artificially synthesized mRNA as described above. Therefore, it is most preferable as a target of the function-suppressing substance in the present invention. Therefore, in a preferred embodiment, a function-suppressing substance that targets OAS3 is adopted. In fact, the functional inhibition of OAS3 dramatically improves the efficiency of the expression of artificially synthesized mRNA (for example, the experimental results shown in the examples below) and its effect is significantly higher than that when OAS1 or OAS2 is targeted. It has been confirmed that it is expensive. The details of OAS are published in a public database, and information necessary for carrying out the present invention (for example, sequence information necessary for designing siRNA in the case of functional inhibition using RNA interference) is available. It can be easily obtained. In the NCBI database, OAS1 is registered as Gene ID: 4938, OAS2 is registered as Gene ID: 4939, and OAS3 is registered as Gene ID: 4940.

Two or more kinds of function-suppressing substances may be used in combination. In this case, the targets, actions, etc. of each function-suppressing substance may be the same, similar, or different. For example, two or more function-suppressing substances selected from the group consisting of a function-suppressing substance of OAS1, a function-suppressing substance of OSA2, and a function-suppressing substance of OAS3 are used in combination. It is also possible to use two or more kinds of function-suppressing substances having the same target in combination. For example, two or more different functional inhibitors targeting OAS3 can be used together.

Examples of the function-suppressing substance of the present invention are as follows.
(a) siRNA targeting 2'-5'-oligoadenylic acid synthase (OAS)
(b) Nucleic acid construct that produces siRNA targeting 2'-5'-oligoadenylic acid synthase (OAS) intracellularly
(c) Antisense nucleic acid targeting 2'-5'-oligoadenylic acid synthase (OAS)
(d) Ribozymes targeting 2'-5'-oligoadenylic acid synthase (OAS)

The above (a) and (b) are compounds used for suppressing expression by so-called RNAi (RNA interference). In other words, if the compound (a) or (b) above is adopted as a function-suppressing substance, RNAi can suppress the expression of OAS. RNAi is a sequence-specific post-transcriptional gene suppression process that can be triggered in eukaryotic cells. RNAi for mammalian cells uses short double-stranded RNA (siRNA) with a sequence corresponding to the sequence of the target mRNA. Usually, siRNA is 21 to 23 base pairs. Mammalian cells are known to have two pathways (sequence-specific and sequence-non-specific pathways) that are affected by double-stranded RNA (dsRNA). In the sequence-specific pathway, relatively long dsRNAs are split into short interfering RNAs (ie siRNAs). On the other hand, the sequence non-specific pathway is considered to be triggered by any dsRNA regardless of the sequence as long as it is longer than a predetermined length. In this pathway, dsRNA becomes two enzymes, the active form, PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2, and 2', 5'oligoadenyl, which is involved in the synthesis of RNase L-activating molecule. Acid synthase is activated. To minimize the progression of this non-specific pathway, it is preferable to use double-stranded RNA (siRNA) shorter than about 30 base pairs (Hunter et al. (1975) J Biol Chem 250: 409-17). See Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8. sea bream).

In order to generate target-specific RNAi, siRNA consisting of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into cells or expressed intracellularly. Just do it. The above (a) is a compound corresponding to the former method, and similarly, the above (b) is a compound corresponding to the latter method.

A siRNA that targets a specific gene is usually a double-stranded RNA that is a hybrid of a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of a complementary sequence thereof. The length of the "continuous region" here is usually 15 to 30 bases, preferably 18 to 23 bases, and more preferably 19 to 21 bases.

It is known that double-stranded RNA having an overhang of several bases at the end exerts a high RNAi effect. Therefore, also in the present invention, it is preferable to adopt siRNA having such a structure. The length of the base forming the overhang is not particularly limited, but is preferably 2 bases (eg, TT, UU, UG).

SiRNA consisting of modified RNA may be used. Examples of modifications here include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).

The dsRNA used in the RNAi method can be prepared in vitro or in vivo by chemical synthesis or using a suitable expression vector. Expression vector methods are particularly effective in preparing relatively long dsRNAs. Sequences unique to the target sequence (continuous sequences) are usually used in the design of siRNA. Programs and algorithms for selecting an appropriate target sequence have been developed.

An example of the siRNA sequence targeting OAS1 is shown below.
CUACAGAGAGACUUCCUGA dTdT (SEQ ID NO: 14. Sequence used in Examples described later)

An example of the siRNA sequence targeting OAS2 is shown below.
CGCUCUGAGCUUAAAUGAU dTdT (SEQ ID NO: 15. Sequence used in Examples below)

An example of the siRNA sequence targeting OAS3 is shown below.
GAAGGAUGCUUUCAGCCUA dTdT (SEQ ID NO: 16. Sequence used in Examples described later)

The "nucleic acid construct that produces siRNA intracellularly" in (b) above refers to a nucleic acid molecule that produces the desired siRNA (siRNA that causes RNAi against the target gene) by the intracellular process when it is introduced into the cell. .. One example of the nucleic acid construct is shRNA (short hairpin RNA). shRNA has a structure in which sense RNA and antisense RNA are linked via a loop structure (hairpin structure), and the loop structure is cleaved in the cell to become double-stranded siRNA, which brings about the RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

Another example of a nucleic acid construct is a vector capable of expressing the desired siRNA. Such vectors are vectors (called stem-loop type or short hairpin type) that express shRNAs that are converted to siRNA by a later process (with a sequence encoding the shRNA inserted), sense RNA and antisense RNA. Can be mentioned as a vector (called a tandem type) that separately expresses. These vectors can be prepared by those skilled in the art according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi. M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et al. (2002) Proc Natl Acad Sci USA 99 (8): 5515-5520; Paddison PJ et al. (2002) Genes Dev. 16: 948-958 etc.). Currently, various RNAi vectors are available. The vector of the present invention may be constructed using such a known vector. In this case, an insert DNA encoding a desired RNA (for example, shRNA) is prepared and then inserted into the cloning site of the vector to obtain an RNAi expression vector. The origin and structure of the vector are not limited as long as it has the function of generating siRNA that exerts RNAi action on the target gene in the cell.

The above (c) is a compound used for suppressing expression by the antisense method. In other words, if the compound of (c) above is adopted as a function-suppressing substance, the expression of OAS can be suppressed by the antisense method. When inhibiting expression by the antisense method, for example, an antisense construct that produces RNA complementary to the unique portion of mRNA encoding OAS when transcribed in the target cell is used. Such antisense constructs are introduced into target cells, for example, in the form of expression plasmids. On the other hand, as an antisense construct, an oligonucleotide probe that hybridizes with an mRNA / or genomic DNA sequence encoding OAS and inhibits its expression when introduced into a target cell can also be adopted. As such oligonucleotide probes, those that are resistant to endogenous nucleases such as exonucleases and / or endonucleases are preferably used.

When a DNA molecule is used as an antisense nucleic acid, an oligodeoxyribonucleotide derived from a region containing a translation initiation site (for example, a region of -10 to +10) of mRNA encoding OAS is preferable.

The complementarity between the antisense nucleic acid and the target nucleic acid is preferably strict, but some mismatch may be present. The ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree and length of complementarity of both nucleic acids. Generally, the longer the antisense nucleic acid used, the more stable double chains (or triple chains) can be formed with the target nucleic acid, even if the number of mismatches is large. One of ordinary skill in the art can use standard techniques to determine the degree of acceptable mismatch.

The antisense nucleic acid may be DNA, RNA, or a chimeric mixture thereof, or a derivative or modified form thereof. Further, it may be single-stranded or double-stranded. By modifying the base portion, the sugar portion, or the phosphate skeleton portion, the stability of the antisense nucleic acid, the hybridization ability, and the like can be improved. In addition, antisense nucleic acids include substances that promote cell membrane transport (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. . 84: 648-652; PCT Publication No. W088 / 09810, published December 15, 1988), or substances that enhance the affinity for specific cells may be added.

The antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems, etc.). For the production of nucleic acid modifiers and derivatives, for example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.

Strong promoters such as pol II and pol III can be utilized to enhance the action of antisense nucleic acids in target cells. That is, if a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, transcription of a sufficient amount of antisense nucleic acid can be ensured by the action of the promoter.

Expression of the antisense nucleic acid can be carried out by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells). For example, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter from the 3'end region of the Raus sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine. -Promoters such as the kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.

In one aspect of the present invention, suppression of expression by ribozyme is utilized (in the case of the compound of (d) above). The target mRNA can also be disrupted with a ribozyme that cleaves the mRNA with a site-specific recognition sequence, but a hammerhead ribozyme is preferred. For example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to for the method of constructing the hammerhead ribozyme.

Ribozymes may be constructed using modified oligonucleotides, for example for the purpose of improving stability and targeting ability, as in the case of utilizing the antisense method. In order to generate an effective amount of ribozyme in the target cell, for example, a nucleic acid construct in which the DNA encoding the ribozyme is placed under the control of a strong promoter (eg, pol II or pol III) can be used. preferable.

Here, it is also possible to use a 2', 5'-oligoadenylic acid (2-5A) derivative as the function-suppressing substance in the present invention. In this case, the 2-5A derivative, which is a function inhibitor, competitively inhibits the binding of 2-5A to the endonuclease RNase L, resulting in inhibition of OAS function. The structure of the 2-5A derivative is not particularly limited as long as it exhibits the property of competitively inhibiting the binding of 2-5A to the endonuclease RNase L. Further, an existing 2-5A derivative may be used, or a newly synthesized 2-5A derivative may be used. It can be used as a function-suppressing substance of the present invention by measuring the amount of binding of 2-5A when the test 2-5A derivative is coexisted under the condition that 2-5A can bind to the endonuclease RNaseL2. It is possible to judge or evaluate whether or not it corresponds to a -5A derivative. Therefore, a person skilled in the art can easily identify a 2-5A derivative that can be used as a function-suppressing substance of the present invention.

The amount of the function-suppressing substance used in the composition of the present invention has a desired effect in the target cell while considering the purpose of use of the composition of the present invention, the characteristics of the function-suppressing substance used, the type of the target cell, and the like. You can set it so that it can be demonstrated. When siRNA is adopted as a function-suppressing substance, for example, it is advisable to contain siRNA in a culture medium so that its concentration is 10 to 50 nM as a single dose.

4. Introduction method In order to express the target gene in the target cell using the present invention, the following steps (1) and (2) are typically performed.
(1) Step of introducing 2'-5'-oligoadenylate synthase function inhibitor (second element of the present invention) into target cells
(2) Step of introducing mRNA of target gene (first element of the present invention) into the target cell

The introduction of siRNA as a function-suppressing substance into a target cell in step (1) and the introduction of mRNA into a target cell in step (2) can be performed by a known method. For example, calcium phosphate co-precipitation, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl). . Acad. Sci. USA 84,7413-7417 (1987)), Microinjection (Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. USA 73,366-370 (1976)), Hanahan's method ( Hanahan, D., J. Mol. Biol. 166, 557-580 (1983)), Lithium acetate method (Schiestl, RH et al., Curr. Genet. 16, 339-346 (1989)), Protoplast-polyethylene glycol Method (Yelton, MM et al., Proc. Natl. Acad. Sci. 81, 1470-1474 (1984)), ultrasonic gene transfer method, method using cationic polyamic acid (for example, JP-A-2011-173802). (See), a method using a polyion complex (PIC) type polymer micelle composed of a block copolymer having a cationic polymer segment and a non-electroporation hydrophilic polymer segment (for example, Japanese Patent Application Laid-Open No. 2004-352972, International Publication). It can be carried out by (see the pamphlet No. 2012/005376) and the like.

As mentioned above, considering the time required for the onset of action of the inhibitory substance, it is preferable to introduce the inhibitory substance and mRNA in this order and at intervals. Therefore, preferably, step (2) is performed when a predetermined time (for example, 1 hour to 48 hours, preferably 4 hours to 30 hours) has elapsed after step (1). However, step (1) and step (2) may be performed at the same time. In this case, for example, a mixture of a function-suppressing substance and mRNA is prepared prior to the introduction operation, and this is introduced into the target cell (in this case, steps (1) and (2) are simultaneously performed as one operation. Will be done). Alternatively, a function-suppressing substance and mRNA are prepared separately, and both are introduced into the target cells under conditions where there is substantially no time difference.

5. Applications According to the composition of the present invention, the expression of artificially synthesized mRNA can be streamlined in the target cell, and the target protein is highly expressed. Therefore, the present invention can be applied to various uses in which high expression of the target protein is desired. Examples of applications of the present invention include (A) various viral diseases (eg, hepatitis B, acquired immunodeficiency syndrome AIDS, adult T-cell leukemia) and genetic diseases (eg, Duchenne muscular dystrophy, cystic adenomyosis, β-salasemia, etc.) Treatment of Hurler syndrome, retinal pigment degeneration, X-chain renal dysfunction), (B) cancer immunotherapy, (C) iPS cell production, (D) pluripotency of stem cells (eg iPS cells and ES cells) Noh stem cells) or induction of differentiation of precursor cells.

(A) and (B) utilize the present invention as a so-called RNA drug. A specific example of (A) is the treatment of hepatitis B. For example, by using an mRNA in which a nuclease (ZFN, TALEN, or CRISPR-Cas9) gene that cleaves and degrades viral DNA integrated into the genome is incorporated as a target gene, the method using a conventional viral vector becomes a problem. Viral treatment without cancer risk becomes possible. As described above, the composition of the present invention is also useful as a virus removing agent. In the treatment of hereditary diseases, for example, a disease-causing gene (a gene that causes a disease due to functional deterioration or deficiency) is used as a target gene, and the present invention is applied. In the use of (B), the present invention is used to introduce an mRNA of a cancer antigen into an antigen-presenting cell to produce a cancer vaccine in the body. If the present invention is applied to the application of (C), it becomes possible to introduce a reprogramming factor without using a viral vector, and thus it is possible to overcome the problem of carcinogenesis of cells. Similar advantages can be obtained in the application of (D).

When the composition of the present invention is used as an RNA drug, it can be formulated according to a conventional method. When formulating, other components that are acceptable for the formulation (eg, buffers, excipients, disintegrants, emulsifiers, suspensions, painkillers, stabilizers, preservatives, preservatives, saline) , Carrier, etc.) can be contained. As the buffering agent, a phosphate buffer solution, a citric acid buffer solution, or the like can be used. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. As the buffer, phosphate, citrate, acetate and the like can be used. As the emulsifier, gum arabic, sodium alginate, tragant and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the pain-relieving agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. Propylene glycol, ascorbic acid and the like can be used as the stabilizer. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited. Examples of dosage forms are injections, tablets, powders, fine granules, granules, capsules and syrups.

The RNA drug of the present invention is targeted by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applies. These routes of administration are not exclusive to each other, and two or more arbitrarily selected routes can be used in combination (for example, intravenous injection or the like at the same time as oral administration or after a predetermined time has elapsed). The "subject" here is not particularly limited, and includes humans and non-human mammals (including pet animals, livestock, and experimental animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, and goats. , Sheep, dogs, cats, chickens, mammals, etc.). In a preferred embodiment, the RNA pharmaceutical of the present invention is applied to humans.

The dose of the RNA drug of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, gender, body weight, etc. are generally taken into consideration. Those skilled in the art can set an appropriate dose in consideration of these matters. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.

1. 1. Purpose The following studies were conducted with the aim of improving the efficiency of expression of artificially synthesized mRNA, which is expected to have various clinical applications, in target cells.

2. 2. Research materials and methods (1) plasmid
The vector pBK-5F-EGFP-pA72 for RNA transfection was prepared as follows. First, oligonucleotides 5'-ACC CTC GAC CCC ACC ATG GCA TCA ATG GAT -3'(SEQ ID NO: 17) and 5'-ATC GAA TTC AAG CTT AGT ACA GCT CGT CCA TGC C -3'(SEQ ID NO: 18). PCMV-5xFlag-EGFP (Hosoda, N., Funakoshi, Y., Hirasawa, M., Yamagishi, R., Asano, Y., Miyagawa, R., Ogami, K., Tsujimoto, M., Hoshino, DNA fragments obtained by the PCR method using S. (2011) Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase. EMBO J 30, 1311-1323.) As a template were treated with XhoI and EcoRI. This DNA fragment was inserted into the XhoI and EcoRI sites of pBluskript II SK (-) (Agilent Technologies, Inc.) to obtain pBK-5F-EGFP. Next, oligonucleotides 5'-CTT GAA TTC GAT ATC GTC GAC GCT CGC TTT CTT GCT GTC CAA TTT CT -3'(SEQ ID NO: 19) and 5'-GTC TCT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTA GAC ATC ATT GCA ATG AAA A -3'(SEQ ID NO: 20) pFlag-CMV5 / TO-BGG (Funakoshi et al., Mechanism of mRNA deadenylation: evidence) DNA fragments obtained by the PCR method using for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases. Genes Dev. 2007 Dec 1; 21 (23): 3135-48.) As a template were treated with EcoRI. This DNA fragment was inserted into the EcoRI site of pBK-5F-EGFP and the blunted BamHI site to prepare pBK-5F-EGFP-pA72.

pCMV-5xMyc-OAS3 was prepared as follows. First, HeLa cells using oligonucleotides 5'-CGG AAG CTT ATG GAC TTG TAC AGC ACC CCG-3'(SEQ ID NO: 21) and 5'-ATC TCA CAC AGC AGC CTT CAC TGG-3' (SEQ ID NO: 22). Using the derived total RNA as a template, after reverse transcription, the DNA fragment obtained by the PCR method was treated with HindIII and EcoRV. This DNA fragment was pCMV-5xMyc (Hosoda, N., Funakoshi, Y., Hirasawa, M., Yamagishi, R., Asano, Y., Miyagawa, R., Ogami, K., Tsujimoto, M., Hoshino, S. (2011) Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase. EMBO J 30, 1311-1323.) Was inserted into the HindIII and EcoRV sites to prepare pCMV-5xMyc-OAS3.

pCMV-5xMyc-PABPC1 was prepared as follows. First, the oligonucleotides 5'-TAG CGG ATC CAT GAA CCC CAG TGC CCC CAG-3'(SEQ ID NO: 23) and 5'-CCC TGA GTC GAC TTA AAC AGT TGG AAC ACC-3' (SEQ ID NO: 24) were used. pGEX6P1-PABP (Hoshino, S., Imai, M., Mizutani, M., Kikuchi, Y., Hanaoka, F., Ui, M., and Katada, T. (1998). Molecular cloning of a novel member of DNA fragments obtained by PCR using the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1. J Biol Chem 273, 22254-22259.) As a template were treated with BamHI and SalI. Smoothed SalI site. This DNA fragment was pCMV-5xMyc (Hosoda, N., Funakoshi, Y., Hirasawa, M., Yamagishi, R., Asano, Y., Miyagawa, R., Ogami, K., Tsujimoto, M., Hoshino, S. (2011) Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase. EMBO J 30, 1311-1323.) Was inserted into the BglII site and the smoothed NotI site to prepare pCMV-5xMyc-PABPC1. ..

pCMV-5xMyc-Dom34 was prepared as follows. First, the oligonucleotides 5'-TCC GAA TTC ATG AAG CTC GTG AGG AAG AAC-3'(SEQ ID NO: 25) and 5'-GGC GAA TTC TTA ATC CTC TTC AGA ACT GGA -3'(SEQ ID NO: 26) were used. pFlag-Dom34 (Saito, S., Hosoda, N., and Hoshino, S. (2013). The Hbs1-Dom34 protein complex functions in non-stop mRNA decay in mammalian cells. J Biol Chem 288, 17832-17843.) The DNA fragment obtained by the PCR method using the above as a template was treated with EcoRI. This DNA fragment was pCMV-5xMyc (Hosoda, N., Funakoshi, Y., Hirasawa, M., Yamagishi, R., Asano, Y., Miyagawa, R., Ogami, K., Tsujimoto, M., Hoshino, S. (2011) Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase. EMBO J 30, 1311-1323.) Was inserted into the EcoRI site to prepare pCMV-5xMyc-Dom34.

pCMV-5xFlag-RNaseL was prepared as follows. First, the oligonucleotides 5'-CTT GAA TTC ATG GAG AGC AGG GAT CAT AAC AAC-3'(SEQ ID NO: 27) and 5'-ATC TCA GCA CCC AGG GCT GGC CAA CCC-3' (SEQ ID NO: 28) were used. Using the total RNA derived from HeLa cells as a template, after reverse transcription, the DNA fragment obtained by the PCR method was treated with EcoRI and EcoRV. This DNA fragment was pCMV-5xFlag (Ruan, L., Osawa, M., Hosoda, N., Imai, S., Machiyama, A., Katada, T., Hoshino, S., and Shimada, I. (2010) ). Quantitative characterization of Tob interactions provides the thermodynamic basis for translation termination-coupled deadenylase regulation. J Biol Chem 285, 27624-27631.) Was inserted into the EcoRI and EcoRV sites to prepare pCMV-5xFlag-RNaseL.

pCMV-5xFlag-RNaseL Y312A was prepared as follows. First, pCMV-5xFlag- using oligonucleotides 5'-GCC GAC CAT TCC CTT GTG AAG GTT-3'(SEQ ID NO: 29) and 5'-ATT CCG CCT CGC TGT CAT AAC AAG-3' (SEQ ID NO: 30). DNA fragments obtained by the inverse-PCR method using RNaseL as a template were ligated to prepare pCMV-5xFlag-RNaseL Y312A.

The sequence of OAS3 is represented by SEQ ID NO: 32 (amino acid sequence) and SEQ ID NO: 33 (base sequence), the sequence of PABPC1 is designated by SEQ ID NO: 34 (amino acid sequence) and SEQ ID NO: 35 (base sequence), and the sequence of Dom34 is designated by SEQ ID NO. The sequence of RNase L is sequenced with SEQ ID NO: 38 (amino acid sequence) and SEQ ID NO: 39 (base sequence), and the sequence of RNase L Y312A is sequenced with SEQ ID NO: 40 (amino acid sequence) in 36 (amino acid sequence) and SEQ ID NO: 37 (base sequence). It is shown in No. 41 (base sequence), respectively.

(2) siRNA sequence <siRNA targeting OAS1>
CUACAGAGAGACUUCCUGA dTdT (SEQ ID NO: 14)
<SiRNA targeting OAS2>
CGCUCUGAGCUUAAAUGAU dTdT (SEQ ID NO: 15)
<SiRNA targeting OAS3>
GAAGGAUGCUUUCAGCCUA dTdT (SEQ ID NO: 16)

(3) RNA synthesis
Using T7 RNA polymerase, 5xFlag-EGFP-pA72 mRNA was synthesized using pBK-5F-EGFP-pA72 treated with BsmBI as a template. RNA synthesis was performed using T7 RNA polymerase (Takara Bio Inc.) according to the instruction manual.

(4) Transfection
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 5% fetal bovine serum at 37 ° C in the presence of 5% CO ₂ . HeLa cells were seeded on a 35 mm dish to 25% confluence, cultured for 24 hours, and then 10 nM or 50 nM siRNA per dish was introduced using Lipofectamine RNAiMAX (Life Technologies) according to the instruction manual. 48 hours after the introduction of siRNA, 1.0 μg of RNA was introduced using Lipofectamine RNAiMAX (Life Technologies) according to the instruction manual. The plasmid was introduced using PEI MAX (Polysciences, Inc) according to the instruction manual.

(5) RNA analysis
Isolation of total RNA from HeLa cells after RNA transfection was performed by a method using guanidine thiocyanate, acidic phenol, and chloroform (AGPC method). The prepared total RNA was separated by agarose MOPS buffer gel (20 mM MOPS (pH 7.0), 5 mM sodium acetate, 1 mM EDTA, 2.0% agarose, 2.46 M formaldehyde) and then under alkaline conditions (3 M sodium chloride, It was transferred to a nylon film Biodyne-B (Pall) with 8 mM sodium hydroxide). Post-transcriptional nylon membranes were UV-fixed and then hybridized overnight with the Flag probe in 7% SDS, 250 mM Na ₃ PO ₄ (pH 7.4), 2 mM EDTA solution. The Flag probe was prepared by Terminal Deoxynucleotidyl Transtransferase (TdT) (Life Technologies) at the 3'end of oligonucleotide 5'-CTT ATC GTC GTC ATC CTT GTA ATC-3'(SEQ ID NO: 31) [α- ³³ ]. It was prepared by incorporating P] dATP. Radioactivity incorporated into the nylon membrane was detected using a bioimaging analyzer Tyhoon FLA 7000 (GE Healthcare).

(6) Protein analysis The intracellular expression of the protein was performed by the Western blotting method shown below. Preparation of protein lysates from cells after introduction is performed with SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 4% glycerol, 2% SDS, 2% 2-mercaptoethanol, 0.004% bromophenol blue). Was used. Protein lysates were separated by SDS-PAGE using 8, 10, 12, or 15% acrylamide and then electrically transferred to a nitrocellulose membrane BioTrace NC (Pall). The nitrocellulose membrane after transcription is an anti-Flag M2 mouse monoclonal antibody (Sigma), an anti-Myc 9E10 mouse monoclonal antibody (Roche), an anti-Myc (A-14) rabbit polyclonal antibody (Santa Cruz), or an anti-GAPDH antibody (Saito, S., Hosoda, N., Hoshino, S. (2013) Hbs1-Dom34 functions in non-stop mRNA decay (NSD) in mammalian cells. J Biol Chem 288, 17832-17843.) And Peroxidase-added anti-mouse IgG (Jackson) ImmunoResearch Laboratories, Inc.), Peroxidase-added anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc.), or Peroxidase-added anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.). The peroxidase enzyme activity on the nitrocellulose membrane was detected by LAS3000mini (Fuji Photo Film Co., Ltd.) using the luminol chemiluminescence method.

(7) Immunoprecipitation method The protein binding experiment was performed by the immunoprecipitation method shown below. After cell recovery, buffer A (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl ₂ , 0.5% Nonidet P-40, 1 mM DTT, 5 mg / ml cOmplete mini (Roche) and 1 μg / ml RNase A (Sigma)) or Buffer B (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 5 mg / ml cOmplete mini (Roche) and 40 U / ml Cells were lysed by RNase inhibitor (TAKARA)). Add anti-Flag M2 agarose (Sigma) or anti-c-Myc agarose affinity gel antibody produced in rabbit (Sigma) to the solubilized fraction obtained by centrifugation at 15000 rpm / 10 minutes, and invert at 10 ° C for 1 hour. It was mixed. Then buffer C (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl ₂ , 0.5% Nonidet P-40, 1 mM DTT, 0.1 mM PMSF, 2 μg / ml aprotinin, 2 μg / ml leupeptin and After washing the beads with 2 μg / ml Pepstatin A) or buffer D (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5% Nonidet P-40 and 1 mM DTT), SDS-PAGE sample buffer was added. Protein was detected by Western blotting and mRNA was detected by Northern blotting.

3. 3. result
Luciferase siRNA was introduced into HeLa cells as OAS1 siRNA, OAS2 siRNA, OAS3 siRNA or control siRNA. 48 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, 3 hours later the cells were washed with PBS and 0, 1, 2 and 4 hours later the cells were harvested. Total RNA was isolated from the collected cells and analyzed by the Northern blot method. As a result, as shown in FIG. 1, stabilization of 5xFlag-EGFP-pA72 mRNA was observed by knockdown of OAS3, but no significant effect was observed in knockdown of OAS1 and OAS2. It was revealed that OAS3 functions in the degradation of artificially synthesized mRNA. This result indicates that inhibition of OAS3 (see FIG. 3), which identifies exogenous artificially synthesized mRNA, stabilizes alien artificially synthesized mRNA.

Luciferase siRNA was introduced into HeLa cells as OAS1 siRNA, OAS2 siRNA, OAS3 siRNA or control siRNA. 48 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, 6 hours later the cells were washed with PBS and 7 hours later the cells were harvested. Lysates of the recovered cells were prepared, and the intracellular expression level of the protein (5xFlag-EGFP) was analyzed by Western blotting. As a result, as shown in FIG. 2, knockdown of OAS3 increased the expression level of 5xFlag-EGFP protein, but knockdown of OAS1 and OAS2 did not significantly increase the expression level. It was clarified that the inhibition of OAS3 function stabilizes exogenous artificially synthesized mRNA and increases the amount of translation. This result indicates that inhibition of OAS3 can increase the amount of protein produced from exogenous artificially synthesized mRNA.

We introduced pCMV-5xMyc-OAS3, pCMV-5xMyc-PABPC1 or pCMV-5xMyc as a control into HeLa cells. 24 hours after introduction, 5xFlag-EGFP-pA72 mRNA was introduced, and 3 hours after that, cells were collected. Lysates of the recovered cells were prepared, and an immunoprecipitation experiment using an anti-c-Myc antibody was performed. The 5xFlag-EGFPpA72 mRNA contained in the lysate and immunoprecipitated fractions was detected by Northern blotting (Fig. 3A upper panel), and the 5xMyc-OAS3 and 5xMyc-PABPC1 proteins were detected by Western blotting (Fig. 3A upper panel). It was shown that OAS3 does not bind to plasmid-derived mRNA, but selectively identifies and binds to foreign artificially synthesized mRNA.

HeLa cells were introduced with pCMV-5xMyc-Dom34 along with pCMV-5xFlag-RNaseL, pCMV-5xFlag-RNaseL Y312A or pCMV-5xFlag as a control. Cells were harvested 24 hours after introduction. Lysates of the recovered cells were prepared, and an immunoprecipitation experiment using an anti-Flag antibody was performed. 5xMyc-Dom34 (Fig. 4A upper panel) and 5xFlag-RNase L or 5xFlag-RNaseL Y312A (Fig. 4A lower panel) contained in the lysate and immunoprecipitation fractions were detected. 5xMyc-Dom34 contained in the immunoprecipitated fraction was quantified (Fig. 4B). A mutation in Y312A that inhibits dimer formation in RNaseL suppressed the binding of RNaseL to Dom34. It was revealed that Dom34 binds more strongly to the activated dimer RNase L.

4. Discussion The results of this study have revealed that OAS3 newly functions for the degradation of artificially synthesized mRNA. As shown in the experiment of FIG. 3, by transfecting the siRNA of OAS3 and suppressing the function of OAS3, it is possible to stabilize the artificially synthesized mRNA in the cell and increase the protein produced. Previously, Dom34 and GTPBP2 have been shown to function in the degradation of artificially synthesized mRNA (Patent Document 1). Furthermore, it has been shown that artificially synthesized mRNA is degraded by the exonuclease-Ski complex and RNaseL that catalyze the degradation in the 3'→ 5'direction (Patent Document 1). Considering this together with these reports, the following molecular mechanism is assumed. OAS3 identifies artificially synthesized mRNA and synthesizes 2-5A. RNase L activated by 2-5A binds Dom34. Dom34 has been reported to bind to stall ribosomes with GTPBP. Dom34, GTPBP and RNaseL are recruited in the vicinity of ribosomes stalled by artificially synthesized mRNA. As a result, artificially synthesized mRNA undergoes endo-cleavage due to the endonuclease activity of RNase L. The resulting fragmented artificially synthesized mRNA is degraded by the exonuclease-Ski complex.

According to the present invention, the expression of artificially synthesized mRNA introduced into a target cell becomes efficient, and high expression of a target gene becomes possible. Applications of the present invention are expected to be RNA drugs (treatment of various viral diseases, cancer immunotherapy, etc.), preparation of iPS cells, and induction of differentiation of stem cells or progenitor cells.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention to the extent that those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

SEQ ID NO: 1: Description of artificial sequence: plasmid sequence SEQ ID NO: 3: Description of artificial sequence: Sequence derived from vector SEQ ID NO: 4: Description of artificial sequence: EGFP
SEQ ID NO: 5: Description of artificial sequence: Restriction enzyme site SEQ ID NO: 14: Description of artificial sequence: siRNA
SEQ ID NO: 15: Description of artificial sequence: siRNA
SEQ ID NO: 16: Description of artificial sequence: siRNA
SEQ ID NOs: 17-30: Description of artificial sequence: Primer SEQ ID NO: 31: Description of artificial sequence: Probe

Claims (10)

The mRNA of the target gene and
2'-5'-combined with a function-suppressing substance of oligoadenylic acid synthase,
The function-suppressing substance is a compound selected from the group consisting of the following (a) to (d).
The 2'-5'-oligoadenylic acid synthase is 2'-5'-oligoadenylate synthase 3.
Composition for expressing the target gene in the target cell:
(a) siRNA targeting 2'-5'-oligoadenylate synthase;
(b) 2'-5'-Nucleic acid construct that produces siRNA targeting oligoadenylate synthase in cells;
(c) Antisense nucleic acid targeting 2'-5'-oligoadenylate synthase;
(d) Ribozyme targeting 2'-5'-oligoadenylic acid synthase. The composition according to claim 1, wherein the kit comprises a first element containing the mRNA and a second element containing the function-suppressing substance. The composition according to claim 1, wherein the composition contains the mRNA and the function-suppressing substance. The composition according to claim 1, wherein the composition contains the mRNA and is introduced in combination with the function-suppressing substance at the time of introduction into a target cell. The composition according to claim 1, which contains the function-suppressing substance and is characterized in that the mRNA is co-introduced at the time of introduction into a target cell. The composition according to any one of claims 1 to 5, wherein the mRNA comprises a 5'cap structure, a Kozak sequence, a coding region of the target gene, and a poly (A) chain. The composition of claim 6, wherein the mRNA further comprises a stabilized cis sequence located between the coding region and the poly (A) chain. The composition according to any one of claims 1 to 7, wherein the gene is an enzyme gene. The composition according to claim 8, wherein the enzyme is a nuclease selected from the group consisting of ZFN, TALEN and CRISPR-Cas9. A hepatitis B virus removing agent containing the composition according to claim 9 as an active ingredient.

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